13C and 15N nuclear magnetic resonance evidence that the active site carboxyl group of dihydrofolate reductase is not involved in the relay of a proton to substrate.

Nuclear magnetic resonance (NMR) spectra for [2-amino,3-15N2]folate and [2-13C]folate complexed with human dihydrofolate reductase, and for complexes of similarly labeled dihydrofolate, show that the N-3 proton of bound folate or dihydrofolate exchanges slowly with solvent and that the bound substrates are in the imino-keto tautomeric form. Previously proposed schemes for substrate protonation that require bound substrate to be in the enolic tautomer are therefore unlikely. The NMR spectra for bound folate are unchanged by raising the pH from 7 to 9.5, whereas those for free folate show marked changes due to ionization for the N-3 proton. The fraction of bound folate with the N-3 proton ionized at pH 9.5 is therefore very small, and the rate constant for the dissociation of the ionized species must be at least 320 times faster than for the protonated species. Comparison of NMR spectra over the pH range 5 to 7 gives no indication of a change in ionization state of the Glu30 carboxyl group over this pH range. This raises doubts about whether the apparent pKa of approximately 6 that describes pH dependence of hydride transfer is due to ionization of this carboxyl group.